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Abstract:

Compositions are disclosed as nucleic acid sequences that may be used as
amplification oligomers, including primers, capture probes for sample
preparation, and detection probes specific for Candida albicans 26S rRNA
sequences or DNA encoding 26S rRNA. Methods are disclosed for detecting
the presence of C. albicans in samples by using the disclosed
compositions in methods that include in vitro nucleic acid amplification
of a 26S rRNA sequence or DNA encoding the 26S rRNA sequence to produce a
detectable amplification product.

Claims:

1. A set of oligomers for use in amplifying a Candida albicans 26S rRNA
sequence or DNA encoding the 26S rRNA sequence, the set of oligomers
comprising first and second amplification oligomers, each of the first
and second amplification oligomers being up to 60 bases in length,
wherein the first amplification oligomer comprises a target binding
region having at least 12 contiguous bases of the base sequence of SEQ ID
NO:19, the DNA equivalent of SEQ ID NO:19, or a combination RNA/DNA
equivalent of SEQ ID NO:19.

2. The set of oligomers of claim 1, wherein the first amplification
oligomer comprises the base sequence of SEQ ID NO:19, the DNA equivalent
of SEQ ID NO:19, or a combination RNA/DNA equivalent of SEQ ID NO:19.

3. The set of oligomers of claim 1, wherein the first amplification
oligomer consists of the base sequence of SEQ ID NO:19, the DNA
equivalent of SEQ ID NO:19, or a combination RNA/DNA equivalent of SEQ ID
NO:19.

4. The set of oligomers of claim 1, wherein the second amplification
oligomer is up to 60 bases in length, and wherein the second
amplification oligomer has at least 12 contiguous bases sequences of the
base sequence of SEQ ID NO:24, the DNA equivalent of SEQ ID NO:24, or a
combination RNA/DNA equivalent of SEQ ID NO:24.

5. The set of oligomers of claim 4, wherein the second amplification
oligomer comprises the base sequence of SEQ ID NO:24, the DNA equivalent
of SEQ ID NO:24, or a combination RNA/DNA equivalent of SEQ ID NO:24.

6. The set of oligomers of claim 5, wherein the first amplification
oligomer comprises the base sequence of SEQ ID NO:19, the DNA equivalent
of SEQ ID NO:19, or a combination RNA/DNA equivalent of SEQ ID NO:19.

7. The set of oligomers of claim 4, wherein the second amplification
oligomer consists of the base sequence of SEQ ID NO:24, the DNA
equivalent of SEQ ID NO:24, or a combination RNA/DNA equivalent of SEQ ID
NO:24 and, optionally, a 5' promoter sequence.

8. The set of oligomers of claim 7, wherein the first amplification
oligomer consists of the base sequence of SEQ ID NO:19, the DNA
equivalent of SEQ ID NO:19, or a combination RNA/DNA equivalent of SEQ ID
NO:19.

9. The set of oligomers of claim 7, wherein the base sequence of the
second amplification oligomer consists of the base sequence of SEQ ID
NO:25.

10. The set of oligomers of claim 9, wherein the first amplification
oligomer consists of the base sequence of SEQ ID NO:19, the DNA
equivalent of SEQ ID NO:19, or a combination RNA/DNA equivalent of SEQ ID
NO:19.

11. The set of oligomers of claim 4 further comprising a probe up to 50
bases in length and comprising a target binding sequence that includes at
least 10 contiguous bases of the base sequence of SEQ ID NO:28, the DNA
equivalent of SEQ ID NO:28, or a combination RNA/DNA equivalent of SEQ ID
NO:28.

12. The set of oligomers of claim 11, wherein the target binding sequence
of the probe comprises the base sequence of SEQ ID NO:28, the DNA
equivalent of SEQ ID NO:28, or a combination RNA/DNA equivalent of SEQ ID
NO:28.

13. The set of oligomers of claim 12, wherein the base sequence of the
probe consists of the base sequence of SEQ ID NO:29, the DNA equivalent
of SEQ ID NO:29, or a combination RNA/DNA equivalent of SEQ ID NO:29.

14. The set of oligomers of claim 11, wherein the target binding sequence
of the probe comprises the base sequence of SEQ ID NO:30, the DNA
equivalent of SEQ ID NO:30, or a combination RNA/DNA equivalent of SEQ ID
NO:30.

15. The set of oligomers of claim 14, wherein the base sequence of the
probe consists of the base sequence of SEQ ID NO:31, the DNA equivalent
of SE ID NO:31, or a combination RNA/DNA equivalent of SEQ ID NO:31.

16. The set of oligomers of claim 11, wherein the probe comprises a label.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of U.S. application Ser. No.
12/715,132, filed Mar. 1, 2010, which is a continuation of U.S.
application Ser. No. 12/539,518, filed Aug. 11, 2009, now U.S. Pat. No.
7,670,780, which is a division of U.S. application Ser. No. 12/335,356,
filed Dec. 15, 2008, now U.S. Pat. No. 7,595,164, which claims the
benefit of U.S. Provisional Application No. 61/016,777, filed Dec. 26,
2007, now abandoned, each of which applications is hereby incorporated by
reference herein in its entirety.

FIELD OF THE INVENTION

[0002]This invention relates to detection of the presence of fungi in a
sample by using molecular biological methods, and specifically relates to
detection of Candida albicans in a sample by amplifying C. albicans
nucleic acid sequences and detecting the amplified nucleic acid
sequences.

SUMMARY

[0003]Disclosed are methods of detecting Candida albicans in a sample,
including environmental samples, biopharmaceutical samples and biological
specimens derived from infected humans, by amplifying and detecting
target sequences contained in C. albicans 26S rRNA or DNA encoding 26S
rRNA. By using specific amplification and detection probe oligomers
disclosed herein, the methods amplify target sequences in 26S rRNA
sequences of C. albicans and detect the amplified products. Some
embodiments monitor the development of specific amplification products
during the amplification step. Some embodiments include a sample
processing step using a capture probe oligomer.

[0004]A method is disclosed for detecting Candida albicans in a sample,
comprising the steps of: mixing a sample that contains a C. albicans
target nucleic acid that is a 26S rRNA sequence or DNA encoding the 26S
rRNA sequence with a first amplification oligomer comprising a target
binding region consisting of SEQ ID NO: 1 or SEQ ID NO: 2 and a second
amplification oligomer comprising a target binding region consisting of
SEQ ID NO: 6; providing an enzyme with nucleic acid polymerase activity
and nucleic acid precursors to make an amplification mixture that
includes the first and second amplification oligomers and the C. albicans
target nucleic acid; elongating in vitro a 3' end of at least one of the
amplification oligomers hybridized to the C. albicans target nucleic acid
by using the enzyme with nucleic acid polymerase activity and the C.
albicans target nucleic acid as a template to produce an amplified
product; and, detecting the amplified product by hybridizing the
amplified product specifically to a detection probe oligomer comprising a
target binding sequence consisting of SEQ ID NO:10 to indicate the
presence Candida albicans in the sample. Some embodiments also include a
sample processing step that captures the C. albicans target nucleic acid
from the sample before the mixing step. The sample processing step may
use a capture probe oligomer that contains a target binding sequence
consisting of SEQ ID NO: 35 or 36, wherein the target binding sequence is
optionally covalently attached to a 3' tail sequence.

[0006]Another method is disclosed for detecting Candida albicans in a
sample, comprising the steps of: mixing a sample that contains a C.
albicans target nucleic acid that is a 26S rRNA sequence or DNA encoding
the 26S rRNA sequence with a first amplification oligomer comprising a
target binding region consisting of SEQ ID NO: 19 and a second
amplification oligomer comprising a target binding region consisting of
SEQ ID NO: 24; providing an enzyme with nucleic acid polymerase activity
and nucleic acid precursors to make an amplification mixture that
includes the first and second amplification oligomers and the C. albicans
target nucleic acid; elongating in vitro a 3' end of at least one of the
amplification oligomers hybridized to the C. albicans target nucleic acid
by using the enzyme with nucleic acid polymerase activity and the C.
albicans target nucleic acid as a template to produce an amplified
product; and, detecting the amplified product by hybridizing the
amplified product specifically to a detection probe oligomer comprising a
target binding sequence consisting of SEQ ID NO:28 to indicate the
presence Candida albicans in the sample. Some embodiments also include a
sample processing step that captures the C. albicans target nucleic acid
from the sample before the mixing step. The sample processing step may
use a capture probe oligomer that contains a target binding sequence
consisting of SEQ ID NO: 35 or 36, wherein the target binding sequence is
optionally covalently attached to a 3' tail sequence.

[0012]Methods are disclosed for sensitively and specifically detecting the
presence of Candida albicans in an environmental, biopharmaceutical or
biological sample by detecting C. albicans nucleic acids. The methods
include performing a nucleic acid amplification of 26S rRNA sequences and
detecting the amplified product, typically by using a nucleic acid probe
that specifically hybridizes to the amplified product to provide a signal
that indicates the presence of C. albicans in the sample. The
amplification step includes contacting the sample with one or more
amplification oligomers specific for a target sequence in 26S rRNA to
produce an amplified product if C. albicans rRNA is present in the
sample. Amplification synthesizes additional copies of the target
sequence or its complement by using at least one nucleic acid polymerase
to extend the sequence from an amplification oligomer (a primer) using a
C. albicans template strand. Preferred embodiments for detecting the
amplified product use a hybridizing step that includes contacting the
amplified product with at least one probe specific for an amplified
sequence, e.g., a sequence contained in the target sequence that is
flanked by a pair of amplification oligomers. The detecting step may be
performed after the amplification reaction is completed, or may be
performed simultaneous with the amplification reaction (sometimes
referred to as "real-time"). In preferred embodiments, the detection step
detects the amplified product using a probe that is detected in a
homogeneous reaction, i.e., detection of the hybridized probe does not
require removal of unhybridized probe from the mixture (e.g., U.S. Pat.
Nos. 5,639,604 and 5,283,174, Arnold Jr. et al.). In preferred
embodiments that detect the amplified product near or at the end of the
amplification step, a linear probe hybridizes to the amplified product to
provide a signal that indicates hybridization of the probe to the
amplified sequence. In preferred embodiments that use real-time
detection, the probe is preferably a hairpin structure probe that
includes a reporter moiety that provides the detected signal when the
probe binds to the amplified product. For example, a hairpin probe may
include a reporter moiety or label, such as a fluorophore ("F"), attached
to one end of the probe and an interacting compound, such as quencher
("Q"), attached to the other end the hairpin structure to inhibit signal
production when the hairpin structure is in the "closed" conformation and
not hybridized to the amplified product, whereas a detectable signal
results when the probe is hybridized to a complementary sequence in the
amplified product, thus converting the probe to a "open" conformation.
Examples of hairpin structure probe include a molecular beacon, molecular
torch, or hybridization switch probe and other forms (e.g., U.S. Pat.
Nos. 5,118,801 and 5,312,728, Lizardi et al; U.S. Pat. Nos. 5,925,517 and
6,150,097, Tyagi et al.; U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274
and 6,361,945, Becker et al.; US Pub. No. 2006-0068417 A1, Becker et al.;
and, US Pub. No. 2006-0194240 A1, Arnold Jr. et al.).

[0013]To aid in understanding this disclosure, some terms used herein are
described below. Unless otherwise described, scientific and technical
terms used herein have the same meaning as commonly understood by those
skilled in the relevant art based on technical literature, e.g., in
Dictionary of Microbiology and Molecular Biology, 2nd ed. (Singleton et
al., 1994, John Wiley & Sons, New York, N.Y.), The Harper Collins
Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York,
N.Y.), or Dorland's Illustrated Medical Dictionary, 30th ed. (2003,
W.B. Saunders, Elsevier Inc., Philadelphia, Pa.). Unless otherwise
described, techniques employed or contemplated herein are standard
methods well known in the art of molecular biology.

[0014]"Sample" includes any specimen that may contain Candida fungi or
components thereof, such as nucleic acids or nucleic acid fragments.
Samples may be obtained from environmental sources, e.g., water, soil,
slurries, debris, biofilms from containers of aqueous fluids, airborne
particles or aerosols, and the like, which may include processed samples,
such as those obtained from passing an environmental sample over or
through a filter, by centrifugation, or by adherence to a medium, matrix,
or support. Samples may also be obtained from any step along a food
supply chain to support food product safety or any step along a
biopharmaceutical process stream to support sterile product development.
"Biological samples" include any tissue or material derived from a living
or dead mammal, including humans, which may contain Candida or target
nucleic acid derived therefrom, e.g., respiratory tissue or exudates such
as bronchoscopy, bronchoalveolar lavage (BAL) or lung biopsy, sputum,
peripheral blood, plasma, serum, lymph node, gastrointestinal tissue,
urine, exudates, or other body fluids. A sample may be treated to
physically or mechanically disrupt aggregates or cells to release
intracellular components, including nucleic acids, into a solution which
may contain other components, such as enzymes, buffers, salts, detergents
and the like.

[0015]"Nucleic acid" refers to a multimeric compound comprising
nucleosides or nucleoside analogs which have nitrogenous heterocyclic
bases, or base analogs, which are linked by phosphodiester bonds or other
linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or
chimeric DNA-RNA polymers, and analogs thereof. A nucleic acid "backbone"
may be made up of a variety of linkages, including one or more of
sugar-phosphodiester linkages, peptide-nucleic acid (PNA) bonds (PCT No.
WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or
combinations thereof. Sugar moieties of the nucleic acid may be either
ribose or deoxyribose, or similar compounds having known substitutions,
e.g., 2' methoxy substitutions and 2' halide substitutions (e.g., 2'-F).
Nitrogenous bases may be conventional bases (A, G, C, T, U), analogs
thereof (e.g., inosine; The Biochemistry of the Nucleic Acids 5-36, Adams
et al., ed., 11th ed., 1992), derivatives of purine or pyrimidine
bases, e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza-
or aza-pyrimidines, pyrimidine bases having substituent groups at the 5
or 6 position, purine bases having an altered or replacement substituent
at the 2, 6 and/or 8 position, such as 2-amino-6-methylaminopurine,
O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,
4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines, and
pyrazolo-compounds, such as unsubstituted or 3-substituted
pyrazolo[3,4-d]pyrimidine (U.S. Pat. Nos. 5,378,825, 6,949,367 and PCT
No. WO 93/13121). Nucleic acids may include "abasic" positions in which
the backbone does not include a nitrogenous base for one or more residues
(U.S. Pat. No. 5,585,481). A nucleic acid may comprise only conventional
sugars, bases, and linkages as found in RNA and DNA, or may include
conventional components and substitutions (e.g., conventional bases
linked by a 2' methoxy backbone, or a nucleic acid including a mixture of
conventional bases and one or more base analogs). Nucleic acids also
include "locked nucleic acids" (LNA), an analogue containing one or more
LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA
mimicking sugar conformation, which enhances hybridization affinity
toward complementary sequences in single-stranded RNA (ssRNA),
single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et
al., 2004, Biochemistry 43(42): 13233-41). Methods for synthesizing
nucleic acids in vitro are well known in the art.

[0016]The interchangeable terms "oligomer" and "oligonucleotide" refer to
a nucleic acid having generally less than 1,000 nucleotides (nt),
including polymers in a range having a lower limit of about 2 nt to 5 nt
and an upper limit of about 500 nt to 900 nt. Preferred oligomers are in
a size range having a lower limit of about 5 nt to 15 nt and an upper
limit of about 50 nt to 600 nt, and particularly preferred embodiments
are in a range having a lower limit of about 10 nt to 20 nt and an upper
limit of about 22 nt to 100 nt. Preferred oligomers are synthesized by
using any well known enzymatic or chemical method and purified by
standard methods, e.g., chromatography.

[0017]An "amplification oligomer" is an oligonucleotide that hybridizes to
a target nucleic acid, or its complement, and participates in a nucleic
acid amplification reaction. An example of an amplification oligomer is a
"primer" that hybridizes to a template nucleic acid and contains a 3'
hydroxyl end that is extended by a polymerase in an amplification
process. Another example is an oligonucleotide that participates in or
facilitates amplification but is not extended by a polymerase, e.g.,
because it has a 3' blocked end. Preferred size ranges for amplification
oligomers include those that are about 10 to about 60 nt long and contain
at least about 10 contiguous bases, and more preferably at least 12
contiguous bases that are complementary to a region of the target nucleic
acid sequence (or its complementary sequence). The contiguous bases are
preferably at least 80%, more preferably at least 90%, and most
preferably about 100% complementary to the target sequence to which the
amplification oligomer binds. An amplification oligomer may optionally
include modified nucleotides or analogs, or optionally an additional
sequence that participates in an amplification reaction but is not
complementary to or contained in the target or template sequence. For
example, a "promoter-primer" is an oligonucleotide that includes a 5'
promoter sequence that is non-complementary to the target nucleic acid
but is adjacent or near to the target complementary sequence of the
primer. Those skilled in the art will understand that an amplification
oligomer that functions as a primer may be modified to include a 5'
promoter sequence, and thus function as a promoter-primer, and a
promoter-primer can function as a primer independent of its promoter
sequence, i.e., the oligonucleotide may be modified by removal of, or
synthesis without, its promoter sequence. An amplification oligomer
referred to as a "promoter-provider" includes a promoter sequence that
serves as a template for polymerization but the oligonucleotide is not
extended from its 3' end which is blocked and, therefore, not available
for extension by polymerase activity.

[0018]"Amplification" refers to any known in vitro procedure for obtaining
multiple copies of a target nucleic acid sequence or fragments thereof,
or its complementary sequence. Amplification of "fragments" refers to
production of an amplified nucleic acid that contains less than the
complete target nucleic acid or its complement, e.g., by using an
amplification oligonucleotide that hybridizes to and initiates
polymerization from an internal position of the target nucleic acid.
Known amplification methods include, for example, replicase-mediated
amplification, the polymerase chain reaction (PCR), ligase chain reaction
(LCR), strand displacement amplification (SDA), and
transcription-mediated or transcription-associated amplification.
Replicase-mediated amplification uses self-replicating RNA molecules, and
a replicase such as Qβ-replicase (e.g., U.S. Pat. No. 4,786,600,
Kramer et al.). PCR amplification uses a DNA polymerase, pairs of
primers, and thermal cycling to synthesize multiple copies of two
complementary strands of a dsDNA or from a cDNA (e.g., U.S. Pat. Nos.
4,683,195, 4,683,202 and 4,800,159, Mullis et al.). LCR amplification
uses four or more different oligonucleotides to amplify a target and its
complementary strand by using multiple cycles of hybridization, ligation,
and denaturation (e.g., U.S. Pat. No. 5,427,930, Birkenmeyer et al.; U.S.
Pat. No. 5,516,663, Backman et SDA uses a primer that contains a
recognition site for a restriction endonuclease and an endonuclease that
nicks one strand of a hemimodified DNA duplex that includes the target
sequence, whereby amplification occurs in a series of primer extension
and strand displacement steps (e.g., U.S. Pat. No. 5,422,252, Walker et
al.; U.S. Pat. No. 5,547,861, Nadeau et al.; U.S. Pat. No. 5,648,211,
Fraiser et al.).

[0019]"Transcription-associated amplification" or "transcription-mediated
amplification" (TMA) refers to any type of nucleic acid amplification
that uses an RNA polymerase to produce multiple RNA transcripts from a
nucleic acid template. These methods generally use an RNA polymerase, a
DNA polymerase, nucleic acid substrates (dNTPs and rNTPs), and a template
complementary oligonucleotide that includes a promoter sequence, and
optionally may include one or more other oligonucleotides. Variations of
transcription-associated amplification are well known in the art (e.g.,
disclosed in detail in U.S. Pat. Nos. 5,399,491 and 5,554,516, Kacian et
al.; U.S. Pat. No. 5,437,990, Burg et al.; PCT Nos. WO 88/01302 and WO
88/10315, Gingeras et al.; U.S. Pat. No. 5,130,238, Malek et al.; U.S.
Pat. Nos. 4,868,105 and 5,124,246, Urdea et al.; PCT No. WO 95/03430,
Ryder et al.; and, US Pub. No. 2006-0046265 A1, Becker et al.). TMA
methods of Kacian et al. and a one primer transcription-associated method
(US Pub. No. 2006-0046265 A1, Becker et al.) are preferred embodiments of
transcription-associated amplification methods for use in detection of
Candida target sequences as described herein. Although preferred
embodiments are illustrated by such amplification reactions, a person of
ordinary skill in the art will appreciated that amplification oligomers
disclosed herein may be readily used in other amplification methods that
extend a sequence from primer(s) by using a polymerase.

[0020]"Probe" refers to a nucleic acid oligomer that hybridizes
specifically to a target sequence in a nucleic acid, preferably in an
amplified nucleic acid, under conditions that allow hybridization to
permit detection of the target sequence or amplified nucleic acid.
Detection may either be direct (i.e., probe hybridized directly to its
target sequence) or indirect (i.e., probe linked to its target via an
intermediate molecular structure). A probe's "target sequence" generally
refers to a subsequence within a larger sequence (e.g., a subset of an
amplified sequence) that hybridizes specifically to at least a portion of
a probe by standard base pairing. A probe may include target-specific
sequence and other sequences that contribute to the probe's
three-dimensional conformation (e.g., described in U.S. Pat. Nos.
5,118,801 and 5,312,728, Lizardi et al; U.S. Pat. Nos. 6,849,412,
6,835,542, 6,534,274 and 6,361,945, Becker et al.; and, US Pub. No.
2006-0068417 A1, Becker et al.).

[0021]By "sufficiently complementary" is meant a contiguous sequence that
is capable of hybridizing to another sequence by hydrogen bonding between
a series of complementary bases, which may be complementary at each
position in the sequence by standard base pairing (e.g., G:C, A:T or A:U
pairing) or may contain one or more positions, including abasic ones,
which are not complementary bases by standard hydrogen bonding.
Contiguous bases are at least 80%, preferably at least 90%, and more
preferably about 100% complementary to a sequence to which an oligomer is
intended to specifically hybridize. Sequences that are "sufficiently
complementary" allow stable hybridization of a nucleic acid oligomer to
its target sequence under the selected hybridization conditions, even if
the sequences are not completely complementary. Appropriate hybridization
conditions are well known in the art, can be predicted readily based on
base sequence composition, or can be determined by using routine testing
(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
§§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly
at §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57).

[0022]"Sample preparation" refers to any steps or methods that prepare a
sample for subsequent amplification and detection of Candida nucleic
acids present in the sample. Sample preparation may include any known
method of concentrating components from a larger sample volume or from a
substantially aqueous mixture, e.g., by filtration or trapping of
airborne particles from an air sample or microbes from a water sample.
Sample preparation may include lysis of cellular components and removal
of debris, e.g., by filtration or centrifugation, and may include use of
nucleic acid oligomers to selectively capture the target nucleic acid
from other sample components.

[0023]A "capture probe" or "capture oligomer" refers to at least one
nucleic acid oligomer that joins a target sequence and an immobilized
oligomer by using base pair hybridization to specifically or
non-specifically capture the target sequence. A preferred capture probe
embodiment includes two binding regions: a target sequence-binding region
and an immobilized probe-binding region, usually on the same oligomer,
although the two regions may be present on different oligomers joined by
one or more linkers. For example, a first oligomer may include the
immobilized probe-binding region and a second oligomer may include the
target sequence-binding region, and the two different oligomers are
joined by a linker that joins the two sequences into a functional unit.
Examples of non-specific target capture are described in U.S. application
Ser. No. 11/832,367, Becker et al.

[0024]An "immobilized probe" or "immobilized nucleic acid" refers to a
nucleic acid that joins, directly or indirectly, a capture oligomer to an
immobilized support. A preferred immobilized probe is an oligomer joined
to a support that facilitates separation of bound target sequence from
unbound material in a sample. Supports may include known materials, such
as matrices and particles free in solution, e.g., made up of
nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,
silane, polypropylene, metal and preferred embodiments are magnetically
attractable particles. Preferred supports are monodisperse magnetic
spheres (e.g., uniform size ±5%), to which an immobilized probe is
joined directly (via covalent linkage, chelation, or ionic interaction),
or indirectly (via one or more linkers), where the linkage or interaction
between the probe and support is stable during hybridization conditions.

[0025]"Separating" or "purifying" means that one or more components of a
mixture, such as a sample, are removed or separated from one or more
other components. Sample components include target nucleic acids in a
generally aqueous mixture (solution phase), which may include cellular
fragments, proteins, carbohydrates, lipids, and other nucleic acids.
Separating or purifying removes at least 70%, preferably at least 80%,
and more preferably about 95% of the target nucleic acid from other
mixture components.

[0026]A "label" refers to a molecular moiety or compound that is detected
or leads to a detectable signal. A label may be joined directly or
indirectly to a nucleic acid probe. Direct labeling can occur through
bonds or interactions that link the label to the probe, including
covalent bonds or non-covalent interactions, e.g., hydrogen bonds,
hydrophobic and ionic interactions, or formation of chelates or
coordination complexes. Indirect labeling can occur through use of a
bridging moiety or linker (e.g., antibody or additional oligomer), which
is either directly or indirectly labeled, and which may amplify the
detectable signal. Labels include any detectable moiety, such as a
radionuclide, ligand (e.g., biotin, avidin), enzyme, enzyme substrate,
reactive group, chromophore (e.g., dye, particle, or bead that imparts
detectable color), luminescent compound (e.g., bioluminescent,
phosphorescent, or chemiluminescent labels), or fluorophore. Preferred
labels include a "homogeneous detectable label" that provides a
detectable signal in a homogeneous reaction in which bound labeled probe
in a mixture exhibits a detectable change that differs from that of
unbound labeled probe, e.g., stability or differential degradation (e.g.,
U.S. Pat. No. 5,283,174, Arnold et al.; U.S. Pat. No. 5,656,207, Woodhead
et al.; U.S. Pat. No. 5,658,737, Nelson at al.). Preferred labels include
chemiluminescent compounds, preferably acridinium ester ("AE") compounds
that include standard AE and derivatives thereof (described in U.S. Pat.
Nos. 5,656,207, 5,658,737 and 5,639,604). Methods of synthesis and
attaching labels to nucleic acids and detecting signals from labels are
well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989), Chpt. 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842,
5,283,174, and 4,581,333).

[0028]Amplification methods that use transcription-mediated amplification
(TMA) include the steps summarized herein (described in detail in U.S.
Pat. Nos. 5,399,491, 5,554,516 and 5,824,518). The target nucleic acid
that contains the sequence to be amplified is provided as single-stranded
nucleic acid (e.g., ssRNA or ssDNA) or made single-stranded by
conventional methods, e.g., temperature and/or chemical melting of
double-stranded nucleic acid to provide a single-stranded target nucleic
acid. A promoter-primer binds specifically to its target sequence in the
target nucleic acid and an enzyme with reverse transcriptase (RT)
activity extends the 3' end of the promoter-primer using the target
strand as a template to make a cDNA of the target sequence, which is in
an RNA:DNA duplex. Enzymatic RNase activity (e.g., RNaseH) digests the
RNA strand of the RNA:DNA duplex and a second primer binds specifically
to its target sequence on the cDNA strand downstream from the
promoter-primer end. The RT synthesizes a new DNA strand by extending the
3' end of the second primer using the first cDNA as a template to make a
dsDNA that contains a functional promoter sequence. An RNA polymerase
specific for the promoter sequence then initiates transcription to
produce multiple RNA transcripts that are, e.g., about 100 to 1000
amplified copies ("amplicons") of the initial target strand in the
reaction. Amplification continues when the second primer binds
specifically to its target sequence in each amplicon and RT makes a DNA
copy from the amplicon RNA template to produce an RNA:DNA duplex. RNase
in the reaction digests the amplicon RNA from the RNA:DNA duplex and the
promoter-primer binds specifically to its complementary sequence in the
newly synthesized DNA. The RT extends the 3' end of the promoter-primer
to create a dsDNA that contains a functional promoter to which the RNA
polymerase binds to transcribe additional amplicons that are
complementary to the initial target strand. These autocatalytic reactions
make more amplicons repeatedly during the complete amplification
reaction, resulting in about a billion-fold amplification of the target
sequence that was present in the sample. The amplified products may be
detected during amplification, i.e., in real-time, or at completion of
the amplification reaction by using a probe that binds specifically to a
target sequence in the amplified products. Signal detected from the bound
probes indicates the presence of the target nucleic acid in the sample.

[0029]Another transcription-associated amplification method summarized
herein uses one primer and one or more additional amplification oligomers
to amplify nucleic acids in vitro by making transcripts (amplicons) that
indicate the presence of the target nucleic acid in a sample (described
in detail in US Pub. No. 2006-0046265 A1, Becker et al.). Briefly, this
single-primer method uses a primer or "priming oligomer", a
"promoter-provider" oligomer that is modified to prevent synthetic
extension from its 3' end (typically, by including a 3'-blocking moiety)
and, optionally, a binding molecule (e.g., a 3'-blocked extender
oligomer) to terminate elongation of a cDNA from the target strand. This
method includes the steps of binding the target RNA that contains the
target sequence with a primer and, optionally, a binding molecule. The
primer hybridizes to the 3' end of the target strand and enzymatic RT
activity initiates primer extension from the 3' end of the primer to
produce a cDNA, to make a duplex of the new strand and the target strand
(RNA:cDNA duplex). When a binding molecule is included in the reaction,
such as a 3'-blocked oligomer, it binds to the target strand next to the
5' end of the target sequence to be amplified. When the primer is
extended by DNA polymerase activity of RT to produce the cDNA strand,
polymerization stops when the primer extension product reaches the
binding molecule on the target strand and, thus, the 3' end of the cDNA
is determined by the position of the binding molecule on the target
strand, making the 3' end of the cDNA complementary to the 5' end of the
target sequence. The RNA:cDNA duplex is separated, e.g., by RNase H
degradation of the RNA strand, or by using conventional strand separation
methods. Then, the promoter-provider oligomer hybridizes to the cDNA
strand near its 3' end. The promoter-provider oligomer includes a 5'
promoter sequence, a 3' region complementary to a sequence in the 3'
region of the cDNA, and a modified 3' end that includes a blocking moiety
to prevent initiation of DNA synthesis from the 3' end of the
promoter-provider oligomer. In the duplex made of the promoter-provider
oligomer and the cDNA strand, the 3'-end of the cDNA is extended by DNA
polymerase activity of the RT enzyme, using the promoter oligomer as a
template to add a promoter sequence to the cDNA, to make a functional
double-stranded promoter. An RNA polymerase specific for the functional
promoter sequence then binds to the promoter and transcribes RNA
transcripts complementary to the cDNA which are substantially identical
to the target region sequence that was amplified from the initial target
strand. The amplified RNA transcripts then serve as substrates in the
amplification process by binding the primer and serving as a template for
further cDNA production. This method ultimately produces many amplicons
from the initial target nucleic acid present in the sample, i.e., it
makes multiple copies of the target sequence. In embodiments of the
method that do not include the binding molecule, the cDNA made from the
primer has an indeterminate 3' end, but the other steps proceed as
described above.

[0030]Detection of the amplified products may be accomplished by a variety
of methods. The amplified nucleic acids may be associated with a surface
to produce a detectable physical change, such as an electrical signal.
Amplified nucleic acids may be concentrated in or on a matrix and
detected by detecting a signal from the concentrated nucleic acid or an
associated dye (e.g., an intercalating agent such as ethidium bromide or
cyber green). Nucleic acids in solution may be detected by detecting an
increased dye association in the solution phase. Preferred embodiments
detect nucleic acid probes that are complementary to a sequence in the
amplified product and form a probe:amplified product complex that
provides a detectable signal (e.g., U.S. Pat. Nos. 5,424,413 and
5,451,503, Hogan et al.; and, U.S. Pat. No. 5,849,481, Urdea et al.).
Directly or indirectly labeled probes that specifically associate with
the amplified product provide a detectable signal to indicate the
presence of the target nucleic acid in the sample. For example, if a
sample contains a target nucleic acid that is Candida albicans 26S rRNA,
the amplified product contains the target sequence in or a complementary
sequence of the C. albicans 26S rRNA, and the probe binds directly or
indirectly to the amplified product's target sequence to produce a signal
that indicates the presence of C. albicans in the sample.

[0031]Preferred probe embodiments that hybridize specifically to the
amplified product sequences may be oligomers of DNA, RNA, or a mixture of
DNA and RNA nucleotides, which may be synthesized with a modified
backbone, e.g., a synthetic oligonucleotide that includes one or more
2'-methoxy substituted RNA groups. Probes for detection of amplified
Candida rRNA sequences may be unlabeled and detected indirectly (e.g., by
binding to another binding partner that is detected) or may be labeled
with a label that results in a detectable signal. Preferred embodiments
include label compounds that emit a detectable light signal, e.g.,
fluorophores or luminescent compounds detected in a homogeneous mixture.
A probe may include more than one label and/or more than one type of
label, or detection may rely on using a mixture of probes in which each
probe is labeled with a compound that produces a detectable signal (e.g.,
U.S. Pat. Nos. 6,180,340 and 6,350,579). Labels may be attached to a
probe by any of a variety of known means, e.g., covalent linkages,
chelation, and ionic interactions, but preferred embodiments covalently
link the label to the oligonucleotide. Probes may be substantially linear
oligonucleotides, i.e., lacking conformations held by intramolecular
bonds, or may be include functional conformational structures, i.e.,
conformations such those found in hairpin structure probes held together
by intramolecular hybridization. Preferred embodiments of linear
oligomers generally include a chemiluminescent label, preferably an AE
compound.

[0032]Hairpin probes are preferably labeled with any of a variety of
different types of interacting labels, in which one interacting member is
usually attached to the 5' end of the probe and the other interacting
member is attached to the 3' end of the probe. Such interacting members
include those often referred to as a reporter dye/quencher pair, a
luminescent/quencher pair, luminescent/adduct pair, Forrester energy
transfer pair, or dye dimer. A luminescent/quencher pair may be made up
of one or more luminescent labels, such as chemiluminescent or
fluorescent labels, and one or more quenchers. In preferred embodiments,
a hairpin probe is labeled at one end with a fluorophore ("F") that
absorbs light at a particular first wavelength or range and emits light
at a second emission wavelength or range and labeled at the other end
with a quencher ("Q") that dampens, partially or completely, signal
emitted from the excited F when Q is in proximity with the fluorophore.
Such a hairpin probe may be referred to as labeled with a
fluorescent/quencher (FIQ) pair. Fluorophores are well known compounds
that include, e.g., acridine, fluorescein, sulforhodamine 101, rhodamine,
5-(2'-aminoethyl)aminoaphthaline-1-sulfonic acid (EDANS), Texas Red,
Eosine, Bodipy and lucifer yellow (Tyagi et al., Nature Biotechnology
16:49-53, 1998). Quenchers are well known and include, e.g.,
4-(4'-dimethyl-amino-phenylaxo)benzoic acid (DABCYL), thallium, cesium,
and p-xylene-bis-pyridinium bromide. Different F/Q combinations are known
and many combinations may function together, e.g., DABCYL with
fluorescein, rhodamine, or EDANS. Other combinations of labels for
hairpin probes include a reporter dye, e.g., FAM®, TET®, JOE®,
VIC® combined with a quencher such as TAMRA® or a non-fluorescent
quencher. A functional F/Q combination may be determined by using routine
testing using known procedures.

[0033]A preferred embodiment of a hairpin probe is a "molecular torch"
that detects an amplified product to indicate the presence of a target
Candida sequence in a sample after the amplification step. A molecular
torch includes: (1) a target detection means that hybridizes to the
target sequence, resulting in an open conformation; (2) a torch closing
means that hybridizes to the target detecting means in the absence of the
target sequence, resulting in a closed conformation; and (3) a joining
means that joins the target detection means and the torch closing means
(described in detail in U.S. Pat. Nos. 6,849,412, 6,835,542, 6,534,274
and 6,361,945). A torch probe in open conformation results in a
detectable signal that indicates the presence of the amplified target
sequence, whereas the closed conformation produces an amount of signal
that is distinguishable from that of the open conformation indicating
that the target sequence is not present. Another preferred hairpin probe
embodiment is a "molecular beacon" that includes a label on one arm of
the hairpin sequence, a quencher on the other arm, and a loop region
joining the two arms (described in detail in U.S. Pat. Nos. 5,118,801 and
5,312,728). Methods for using such hairpin probes are well known in the
art.

[0034]Oligomers that are not extended by a nucleic acid polymerase include
a blocker group that replaces the 3' OH to prevent enzyme-mediated
extension of the oligomer in an amplification reaction. Blocked
amplification oligomers and/or blocked detection probes present during
amplification (for real-time detection) preferably lack a 3' OH but
include one or more blocking groups located at or near the 3' end. A
blocking group is covalently attached to the 3' terminus of the
oligonucleotide or is located near the 3' end, preferably within five
residues of the 3' end, and is sufficiently large to limit binding of a
polymerase to the oligomer. Many different chemical groups may be used as
a blocking moiety, e.g., alkyl groups, non-nucleotide linkers,
alkane-diol dideoxynucleotides, and cordycepin.

[0035]A preferred method for detection of Candida albicans sequences uses
a transcription-associated amplification with a hairpin probe, e.g.
molecular torch or molecular beacon, because the probe may be added
before amplification, and detection is carried out without further
addition of reagents. For example, a probe may be designed so that the
Tm of the hybridized arms of the hairpin probe (e.g., target binding
domain:target closing domain complex of a molecular torch) is higher than
the amplification reaction temperature to prevent the probe from
prematurely binding to amplified target sequences. After an interval of
amplification, the mixture is heated to open the torch probe arms and
allow the target binding domain to hybridize to its target sequence in
the amplified product. The solution is then cooled to close probes not
bound to amplified products, which closes the label/quencher (F/Q) pair,
allowing detection of signals from probes hybridized to the amplified
target sequences in a homogeneous reaction. For example, the mixture
containing the F/Q labeled hairpin probe is irradiated with the
appropriate excitation light and the emission signal is measured.

[0036]In other embodiments, the hairpin detection probe is designed so
that amplified products preferentially hybridize to the target binding
domain of the probe during amplification, thereby changing the hairpin
from its closed to open conformation as amplification progresses. The
amplification reaction mixture is irradiated at intervals during the
amplification reaction to detect the emitted signal from the open probes
during amplification, i.e., in real-time.

[0037]Preparation of samples for amplification of Candida sequences may
include separating and/or concentrating organisms contained in a sample
from other sample components, e.g., filtration of particulate matter from
air, water or other types of samples. Sample preparation may also include
chemical, mechanical, and/or enzymatic disruption of cells to release
intracellular contents, including Candida 26S rRNA or DNA encoding the
26S rRNA. Sample preparation may include a step of target capture to
specifically or non-specifically separate the target nucleic acids from
other sample components. Non-specific target preparation methods may
selectively precipitate nucleic acids from a substantially aqueous
mixture, adhere nucleic acids to a support that is washed to remove other
sample components, or use other means to physically separate nucleic
acids, including Candida nucleic acid, from a mixture that contains other
components. Other non-specific target preparation methods may selectively
separate RNA, including Candida 26S rRNA, from DNA in a sample.

[0038]In a preferred embodiment, Candida 26S rRNA or DNA encoding 26S rRNA
are selectively separated from other sample components by specifically
hybridizing the Candida nucleic acid to a capture oligomer specific for
the Candida target sequence to form a target sequence:capture probe
complex that is separated from sample components. A preferred embodiment
of specific target capture binds the Candida target:capture probe complex
to an immobilized probe to form a target:capture probe:immobilized probe
complex that is separated from the sample and, optionally, washed to
remove non-target sample components. The capture probe includes a
sequence that specifically binds to the Candida target sequence in 26S
rRNA or in DNA encoding 26S rRNA and also includes a specific binding
partner that attaches the capture probe with its bound target sequence to
a support (e.g., matrix or particle), which facilitates separating the
target sequence from the sample components. In a preferred embodiment,
the specific binding partner of the capture probe is a 3' tail sequence
that is not complementary to the Candida target sequence but that
hybridizes to a complementary sequence on an immobilized probe attached
to the support. Preferred 3' tail sequences are substantially
homopolymeric 10 to 40 nt sequences (e.g., A10 to A40) that
bind to a complementary immobilized sequence (e.g., poly-T) attached to
the support. Target capture occurs in a solution phase mixture that
contains capture oligomers that hybridize specifically to the Candida
target nucleic acid under hybridizing conditions, usually at a
temperature higher than the Tm of the tail sequence:immobilized
probe sequence duplex. The Candida target:capture probe complex is
captured by adjusting the hybridization conditions so that the capture
probe tail then hybridizes to the immobilized probe, and the entire
complex on the support is separated from the other sample components. The
support with the attached complex that includes the Candida target
sequence may be washed to further remove other sample components.
Preferred supports are particulate, such as paramagnetic beads, so that
particles with the complex that includes the captured Candida target
sequence may be suspended in a washing solution and retrieved from the
washing solution by using magnetic attraction. In other embodiments, the
capture probe may bind nonspecifically to nucleic acids in the sample,
including the Candida target sequence, and then similar steps of
attaching the capture probe:nucleic acid complexes to a support and
separating the captured complexes on the support are performed. Whether
target capture is specific or non-specific for the Candida target
sequence, the captured nucleic acids are then subjected to in vitro
amplification specific for the intended Candida target sequence. To limit
the number of handling steps, Candida target nucleic acid may be
amplified by mixing the Candida target sequence in the captured complex
on the support with amplification reagents, or a primer may be included
in the target capture reaction mixture, thus allowing the Candida
specific primer and target sequences to hybridize during target capture
and be separated together from the sample in the captured complex.

[0039]Assays for detection of Candida nucleic acid may optionally include
a non-Candida internal control (IC) nucleic acid that is amplified and
detected in the same assay reaction mixtures by using amplification and
detection oligomers specific for the IC sequence. Amplification and
detection of a signal from the amplified IC sequence demonstrates that
the assay reagents, conditions, and procedural steps were properly used
and performed in the assay if no signal is obtained for the intended
target Candida nucleic acid (e.g., samples that provide negative results
for C. albicans). The IC may be used as an internal calibrator for the
assay when a quantitative result is desired, i.e., the signal obtained
from the IC amplification and detection is used to set a parameter used
in an algorithm for quantitating the amount of Candida nucleic acid in a
sample based on the signal obtained for amplified an Candida target
sequence. A preferred IC embodiment is a randomized sequence that has
been derived from a naturally occurring source (e.g., an HIV sequence
that has been rearranged in a random manner). A preferred IC may be an
RNA transcript isolated from a naturally occurring source or synthesized
in vitro, such as by making transcripts from a cloned randomized sequence
such that the number of copies of IC included in an assay may be
accurately determined. The primers and probe for the IC target sequence
are designed and synthesized by using any well known method provided that
the primers and probe function for amplification of the IC target
sequence and detection of the amplified IC sequence using substantially
the same assay conditions used to amplify and detect the Candida target
sequence and the IC components in the assay do not interfere with those
used to amplify and detect the Candida target sequence. In preferred
embodiments that include a target capture-based purification step, a
target capture probe specific for the IC target is included in the target
capture step so that the IC is treated in the same conditions as used for
the intended Candida analyte in all of the assay steps.

EXAMPLES

[0040]For amplification and detection of target sequences in 26S rRNA
sequences (which include 26S rRNA and DNA encoding 26S rRNA) of Candida
albicans, oligomers were designed that act as amplification oligomers and
detection probes by comparing known sequences of 26S rRNA or gene
sequences encoding 26S rRNA and selecting sequences that are common to C.
albicans isolates, but preferably are not completely identical to 26S
rRNA sequences of other Candida species or other eukaryotic organisms.
Sequence comparisons were conducted by using known 26S rRNA sequences
(rRNA or genes) of Candida species (C. dubliniensis, C. tropicalis and C.
glabrata) and of other fungal species (S. cerevisiae, S. barnettii, S.
exiguus, S. spencerorum, K. lodderae, S. rosinii, S. unisporus, S.
servazzil, K. africanus, S. dairenensis, S. castellii, S. paradoxus, S.
bayanus, K. yarrowii, K polysporus, E. gossypil and E. fibuliger).
Specific oligonucleotide sequences were selected, synthesized in vitro
and characterized with purified rRNA from fungi using standard laboratory
methods. The selected oligomers were further tested by using different
combinations of the amplification oligomers in amplification reactions
with whole cell lysates or total RNA purified from fungi grown in
culture, to determine the relative efficiencies of amplification of the
target sequences by using the selected amplification oligomers. The
efficiencies of different combinations of oligomers were monitored by
detecting the amplified products of the amplification reactions;
generally by binding a labeled probe oligomer to the amplified products
and detecting a signal that indicated the presence of amplified product.

[0041]Preferred embodiments of the selected amplification oligomers for C.
albicans 26S rRNA target sequences are shown in Table 1. Amplification
oligomers include those that may function as primers, promoter-primers,
and/or promoter-provider oligomers. For the latter two, promoter
sequences are shown in lower case in Table 1. Some oligomer embodiments
include only the target-specific sequence of a corresponding
promoter-primer or promoter-provider oligomer, e.g., SEQ ID NO: 4 is a
target-specific sequence that is identical to the target-specific
sequence contained in SEQ ID NO: 5, which includes a 5' promoter
sequence. Those skilled in the art will appreciate that the
target-specific sequences listed in Table may optionally be attached to
the 3' end of any known promoter sequence to function as a
promoter-primer or promoter-provider with the appropriate RNA polymerase
for the chosen promoter sequence. An example of a promoter sequence
specific for the RNA polymerase of bacteriophage T7 is SEQ ID NO: 37
(AATTTAATACGACTCACTATAGGGAGA). Preferred embodiments of amplification
oligomers may include a mixture of DNA and RNA bases, and 2' methoxy RNA
groups, e.g., oligomers of SEQ ID NOs: 1-3 and 19-21 may include RNA
bases and 2' methoxy linkages at the first four positions from the 5'
end. Embodiments of amplification oligomers may be modified by
synthesizing the oligomer with the 3' end blocked to make the oligomer
optimal for functioning as a blocking molecule or promoter-provider
oligomer in a single-primer transcription-associated amplification
reaction. Preferred embodiments of 3'-blocked oligomers include those of
SEQ ID NOs: 5, 7, 9, 16-18, 23, 25, 27 and 32-34 that include a blocked C
near or at the 3' end.

[0042]Preferred embodiments of the selected detection probe oligomers for
detecting amplified products of 26S rRNA sequences or DNA encoding 26S
rRNA are shown in Table 2. Preferred detection probe embodiments are
oligomers that form hairpin configurations by intramolecular
hybridization of the probe sequence, of which preferred embodiments are
those of SEQ ID NOs: 11, 13, 15, 29 and 31. Preferred hairpin probe
oligomers are synthesized with a fluorescent label attached at one end
and a quencher compound attached at the other end of the sequence.
Embodiments of hairpin probes may be labeled with a 5' fluorophore and a
3' quencher, e.g., 5' fluoroscein label with 3' DABCYL quencher. Some
embodiments of hairpin oligomers include a non-nucleotide linker moiety
at selected positions within the sequence, e.g., oligomers that include
an abasic 9-carbon ("C9") linker located in: SEQ ID NO: 11 between nt 5
and nt 6, SEQ ID NO: 13 between nt 20 and nt 21, SEQ ID NO: 15 between nt
17 and nt 18, SEQ ID NO: 29 between nt 16 and nt 17, and SEQ ID NO: 31
between nt 20 and nt 21.

[0043]Embodiments of non-specific and specific capture probe oligomers for
use in sample preparation to separate Candida 26S rRNA target nucleic
acids from other sample components include those that contain the
sequences of SEQ ID NO: 35 (kkkkkkkkkkkkkkkkkk) and SEQ ID NO: 36
(CGAGGCAUUUGGCUACCUUAAGAG), respectively. Preferred embodiments of the
capture probes include a 3' tail region covalently attached to the
sequence to serve as a binding partner that binds a hybridization complex
made up of the target nucleic acid and the capture probe to an
immobilized probe on a support. Preferred embodiments of capture probes
that include the sequences of SEQ ID NOs: 35 and 36 further include 3'
tail regions made up of substantially homopolymeric sequences, e.g., a
dT3A30 sequence.

[0045]Different amplification oligomer combinations were made from those
listed in Table 1 and were tested in single-primer
transcription-associated amplifications as described above, using whole
cell lysates or ribosomal RNA isolated from C. albicans and other fungi
as target nucleic acid. Amplified products were detected by using hairpin
probes (molecular torches) from those listed in Table 2 labeled with a
fluorophore (5' fluorescein) and 3' quencher (DABCYL), detecting the
fluorescence emitted when the probe bound to amplified sequences.

Example 1

Amplification and Detection Probe Oligomer Combinations

[0046]Amplification and detection of a C. albicans 26S rRNA target
sequence was demonstrated in real-time by using a probe that hybridizes
to the amplified product during the amplification reaction. Amplification
was performed by using a single-primer transcription-associated
amplification procedure substantially as described in detail in US Pub.
No. 2006-0046265 A1, conducted by using some of the selected
amplification oligomers. Each of the assays was performed in an
amplification reaction (0.040 ml total volume) that contained the C.
albicans target RNA and amplification reagents substantially as described
for TMA reactions but with a promoter-provider oligomer (12 pmol per
reaction), a primer oligomer (6 pmol per reaction), a blocker oligomer
(0.5 pmol per reaction), and a hairpin probe (molecular torch at 6 pmol
per reaction). Reaction mixtures containing the amplification oligomers,
target and amplification reagents (but not enzymes) were covered to
prevent evaporation, incubated 10 min at 60° C., then 5 min at
42° C., then enzymes were added (10 μl vol) and the reactions
were mixed and incubated for 50 min at 42° C., measuring
fluorescence every 20 sec during the amplification reaction after enzyme
addition.